Connexins are integral membrane proteins that form the gap-junctional channels that mediate cell-to- cell permeation of ions and hydrophilic molecules of Mr <1,000, hence underlying electrical and chemical coupling between neighboring cells. Connexins are essential for embryonic development and normal function of cells and tissues, and they also participate in pathological processes, both genetic and acquired. Six connexin monomers, containing four transmembrane a helices each (M1 to M4), form a gap-junctional hemichannel. Gap-junctional channels are formed by end-to-end docking of gap- junctional hemichannels, one from each of two adjacent cells. The available structural information is insufficient to identify individual transmembrane helices, and therefore we do not know which helices form the pore and how the helices fold in the gap-junctional channels and hemichannels. This proposal aims to address these gaps in knowledge. Our general goal is to understand the structural bases for the permeability properties of the gap-junctional channel and hemichannel pore, and our central hypothesis is that transmembrane helices M1 and M3 line the pore, but the folding of the transmembrane helices is different from that proposed in the current models. Our specific aims are: 1) to determine the accessibility of Cx43 transmembrane-helix residues to the aqueous environment of the pore, 2) to identify the individual Cx43 helices in gap-junctional hemichannels by measuring inter-helix distances, and 3) to determine whether the folding of transmembrane helices within hemichannels formed connexins that display significant differences in molecular size, amino-acid sequence and domain structure (Cx43 and Cx26) are the same. To accomplish these aims, we will employ biochemical, cell- biological and biophysical techniques, including the use of a newly-developed experimental system where we can measure inter-helical distances using luminescence resonance energy transfer in gap- junctional hemichannels containing a single donor and a known number of acceptors, at selected positions. Our proposal will result in the best available gap-junctional hemichannel model. Significance: connexins are essential for the normal development of many organs, including the heart, and mutations of connexins cause a number of genetic diseases, including deafness. Elucidation of the architecture of gap-junctional channel and hemichannel pores is necessary to understand the mechanisms of disease due to connexin mutations.